147 IDENTIFICATION OF PRIMORDIAL GERM CELLS IN PORCINE EMBRYOS FROM THE PRIMITIVE STREAK STAGE

In embryonic stem cell research, Oct-4 is one of the most widely used markers of pluripotency. Moreover, at least in the mouse, this marker is restricted to primordial germ cells (PGCs) after gastrulation. Vimentin is often used as a marker of mesoderm/mesenchyme in embryonic tissues and appears to localize to the same embryonic cells as Oct-4, at least in the bovine epiblast. The expression of neither of these markers has been completely addressed in the pig. Therefore, the purpose of the present study was to examine the expression of Oct-4 and vimentin in the porcine epiblast during differentiation and establishment of the three germ layers, i.e. the process of gastrulation. A total of 410 porcine embryos were collected at 8 to 17 days post-insemination from 29 sows of the Danish Landrace breed. Embryos were categorized based on stereo-microscopic observations into the following stages: pre-streak stages 1 and 2, primitive streak stage, neural groove stage, and somite stage. Specimens were fixed at all stages, dehydrated and embedded in paraffin wax. Selected embryos at each stage (n = 28) were completely cut into serial sections for immunohistochemical evaluation of Oct-4 and vimentin. Pre-streak stage 1 embryos were defined by lack of polarization of the embryonic disk, whereas in pre-streak stage 2 embryos a crescent shaped thickening was seen at the posterior pole of the disk. This thickening, marking the first morphological anterior-posterior polarization of the embryo proper, was shown to be a site of incipient ingression of cells from the epiblast. Immunohistochemical analyses localized Oct-4 to nuclei and vimentin to cytoplasm of both founding and ingressing epiblast cells. During formation of mesoderm and endoderm, at the primitive streak stage, solitary Oct-4 positive cells, i.e. potential PGCs, were seen scattered in the endoderm. Cells of the epiblast displayed positive labeling for Oct-4 until specification for the ectoderm cell lineage at the subsequent neural groove stage. In mesoderm, Oct-4 likewise disappeared by the time of formation of the first somites, defining the following somite stage. Thus, at this stage the only cells labeled for Oct-4, i.e. potential PGCs, were seen solitarily scattered in the endoderm. By the 15-somite stage, such cells were no longer visible in the endoderm but were seen located in the mesoderm, spreading from the stalk of the yolk sac and allantois and extending through the mid- and hindgut areas into the incipient genital ridge. Vimentin localized to the mesenchyme and most other derivatives of neural crest and mesodermal origin. In conclusion, based on Oct-4 labeling and distribution pattern, we strongly believe that we have identified the porcine PGCs from the primitive streak stage.

Download Full-text

X-chromosome activity of the mouse primordial germ cells revealed by the expression of an X-linked lacZ transgene

Development ◽

10.1242/dev.120.10.2925 ◽

1994 ◽

Vol 120 (10) ◽

pp. 2925-2932 ◽

Cited By ~ 6

Author(s):

P.P. Tam ◽

S.X. Zhou ◽

S.S. Tan

Keyword(s):

X Chromosome ◽

Germ Cells ◽

Primordial Germ Cells ◽

Cell Lineage ◽

Primitive Streak ◽

X Inactivation ◽

Lacz Gene ◽

Genital Ridge ◽

X Chromosomes ◽

Fetal Ovary

We have determined the timing of the inactivation and reactivation of the X chromosome in the mouse primordial germ cells (PGCs) by monitoring the expression of an X-linked HMG-lacZ reporter gene. PGCs were identified by their distinct alkaline phosphatase activity and they were first localised in the primitive streak and allantoic bud of the 7.5-day gastrulating embryo. Although inactivation of the transgene was found in some PGCs at these sites, at least 85% of the population were still expressing the lacZ gene. This suggests that, although X-inactivation has commenced during gastrulation, the majority of PGCs still possess two active X chromosomes. Transgene activity remained unchanged during the relocation of PGCs to the hindgut endoderm, but decreased abruptly when PGCs left the hindgut and migrated through the mesentery. X-inactivation was completed during the migration of PGCs, but not simultaneously for the whole population. The first wave of PGCs entering the genital ridge at 9.5 days did not immediately re-activate the silent transgene until about 24 hours later. Re-activation of the transgene took place in over 80% of PGCs entering the genital ridge at 10.5-13.5 days p.c., preceding the entry into meiosis. About 90% of the meiotic germ cells in the 14.5-15.5 day fetal ovary expressed the transgene. Similar profiles of transgene activity were observed in PGCs of embryos that have inherited the lacZ transgene from different parents, showing unequivocally that X-inactivation in the germ cell lineage is not related to parental legacy.(ABSTRACT TRUNCATED AT 250 WORDS)

Download Full-text

Formative pluripotent stem cells show features of epiblast cells poised for gastrulation

Cell Research ◽

10.1038/s41422-021-00477-x ◽

2021 ◽

Author(s):

Xiaoxiao Wang ◽

Yunlong Xiang ◽

Yang Yu ◽

Ran Wang ◽

Yu Zhang ◽

...

Keyword(s):

Stem Cells ◽

Germ Cells ◽

Pluripotent Stem Cells ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

Large Set ◽

Mouse Escs ◽

Epiblast Stem Cells ◽

Early Gastrula

AbstractThe pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

Download Full-text

Primordial germ cells in normal and transected duck blastoderms

Development ◽

10.1242/dev.20.3.247 ◽

1968 ◽

Vol 20 (3) ◽

pp. 247-260

Author(s):

Teresa Rogulska

Keyword(s):

Chick Embryo ◽

Blood Vessels ◽

Germ Cells ◽

Anterior Part ◽

Primordial Germ Cells ◽

Primitive Streak ◽

Experimental Investigations ◽

Suggestive Evidence ◽

Area Pellucida

Suggestive evidence for the extragonadal origin of germ cells in birds was first presented by Swift (1914), who described primordial germ cells in the chick embryo at as early a stage as the primitive streak. According to Swift, primordial germ cells are originally located extra-embryonically in the anterior part of the blastoderm and occupy a crescent-shaped region (‘germinal crescent’) on the boundary between area opaca and area pellucida. Swift also found that primordial germ cells later enter into the blood vessels, circulate together with the blood throughout the whole blastoderm and finally penetrate into the genital ridges, where they become definitive germ cells. Swift's views have been confirmed in numerous descriptive and experimental investigations. Among the latter, the publications of Willier (1937), Simon (1960) and Dubois (1964a, b, 1965a, b, 1966) merit special attention. Dubois finally proved that the genital ridges exert a strong chemotactic influence on the primordial germ cells.

Download Full-text

Dual role of Ovol2 on the germ cell lineage segregation during gastrulation in mouse embryogenesis

Development ◽

10.1242/dev.200319 ◽

2022 ◽

Author(s):

Yuki Naitou ◽

Go Nagamatsu ◽

Nobuhiko Hamazaki ◽

Kenjiro Shirane ◽

Masafumi Hayashi ◽

...

Keyword(s):

Germ Cells ◽

Splice Variant ◽

Epithelial To Mesenchymal Transition ◽

Functional Relationship ◽

Germ Line ◽

Primordial Germ Cells ◽

Cell Lineage ◽

Animal Kingdom ◽

Mesenchymal Transition

In mammals, primordial germ cells (PGCs), the origin of the germ line, are specified from the epiblast at the posterior region where gastrulation simultaneously occurs, yet the functional relationship between PGC specification and gastrulation remains unclear. Here, we show that Ovol2, a transcription factor conserved across the animal kingdom, balances these major developmental processes by repressing the epithelial-to-mesenchymal transition (EMT) driving gastrulation and the upregulation of genes associated with PGC specification. Ovol2a, a splice variant encoding a repressor domain, directly regulates EMT-related genes and consequently induces re-acquisition of potential pluripotency during PGC specification, whereas Ovol2b, another splice variant missing the repressor domain, directly upregulates genes associated with PGC specification. Taken together, these results elucidate the molecular mechanism underlying allocation of the germ line among epiblast cells differentiating into somatic cells through gastrulation.

Download Full-text

Ligand–Receptor Interactions Elucidate Sex-Specific Pathways in the Trajectory From Primordial Germ Cells to Gonia During Human Development

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.661243 ◽

2021 ◽

Vol 9 ◽

Author(s):

Arend W. Overeem ◽

Yolanda W. Chang ◽

Jeroen Spruit ◽

Celine M. Roelse ◽

Susana M. Chuva De Sousa Lopes

Keyword(s):

Germ Cell ◽

Signaling Pathways ◽

Germ Cells ◽

Tyrosine Kinases ◽

Intracellular Localization ◽

Primordial Germ Cells ◽

Cell Lineage ◽

Systematic Analysis ◽

Receptor Interactions

The human germ cell lineage originates from primordial germ cells (PGCs), which are specified at approximately the third week of development. Our understanding of the signaling pathways that control this event has significantly increased in recent years and that has enabled the generation of PGC-like cells (PGCLCs) from pluripotent stem cells in vitro. However, the signaling pathways that drive the transition of PGCs into gonia (prospermatogonia in males or premeiotic oogonia in females) remain unclear, and we are presently unable to mimic this step in vitro in the absence of gonadal tissue. Therefore, we have analyzed single-cell transcriptomics data of human fetal gonads to map the molecular interactions during the sex-specific transition from PGCs to gonia. The CellPhoneDB algorithm was used to identify significant ligand–receptor interactions between germ cells and their sex-specific neighboring gonadal somatic cells, focusing on four major signaling pathways WNT, NOTCH, TGFβ/BMP, and receptor tyrosine kinases (RTK). Subsequently, the expression and intracellular localization of key effectors for these pathways were validated in human fetal gonads by immunostaining. This approach provided a systematic analysis of the signaling environment in developing human gonads and revealed sex-specific signaling pathways during human premeiotic germ cell development. This work serves as a foundation to understand the transition from PGCs to premeiotic oogonia or prospermatogonia and identifies sex-specific signaling pathways that are of interest in the step-by-step reconstitution of human gametogenesis in vitro.

Download Full-text

Identification of tissues and patterning events required for distinct steps in early migration of zebrafish primordial germ cells

Development ◽

10.1242/dev.126.23.5295 ◽

1999 ◽

Vol 126 (23) ◽

pp. 5295-5307 ◽

Cited By ~ 5

Author(s):

G. Weidinger ◽

U. Wolke ◽

M. Koprunner ◽

M. Klinger ◽

E. Raz

Keyword(s):

Germ Cells ◽

Primordial Germ Cells ◽

Cell Lineage ◽

Paraxial Mesoderm ◽

Molecular Characteristics ◽

Dorsoventral Patterning ◽

Wild Type ◽

Migration Process ◽

Early Migration ◽

Somatic Tissues

In many organisms, the primordial germ cells have to migrate from the position where they are specified towards the developing gonad where they generate gametes. Extensive studies of the migration of primordial germ cells in Drosophila, mouse, chick and Xenopus have identified somatic tissues important for this process and demonstrated a role for specific molecules in directing the cells towards their target. In zebrafish, a unique situation is found in that the primordial germ cells, as marked by expression of vasa mRNA, are specified in random positions relative to the future embryonic axis. Hence, the migrating cells have to navigate towards their destination from various starting positions that differ among individual embryos. Here, we present a detailed description of the migration of the primordial germ cells during the first 24 hours of wild-type zebrafish embryonic development. We define six distinct steps of migration bringing the primordial germ cells from their random positions before gastrulation to form two cell clusters on either side of the midline by the end of the first day of development. To obtain information on the origin of the positional cues provided to the germ cells by somatic tissues during their migration, we analyzed the migration pattern in mutants, including spadetail, swirl, chordino, floating head, cloche, knypek and no isthmus. In mutants with defects in axial structures, paraxial mesoderm or dorsoventral patterning, we find that certain steps of the migration process are specifically affected. We show that the paraxial mesoderm is important for providing proper anteroposterior information to the migrating primordial germ cells and that these cells can respond to changes in the global dorsoventral coordinates. In certain mutants, we observe accumulation of ectopic cells in different regions of the embryo. These ectopic cells can retain both morphological and molecular characteristics of primordial germ cells, suggesting that, in zebrafish at the early stages tested, the vasa-expressing cells are committed to the germ cell lineage.

Download Full-text

The use of ‘normal’ and ‘transformed’ gynandromorphs in mapping the primordial germ cells and the gonadal mesoderm in Drosophila

Development ◽

10.1242/dev.35.3.607 ◽

1976 ◽

Vol 35 (3) ◽

pp. 607-616

Author(s):

W. J. Gehring ◽

E. Wieschaus ◽

M. Holliger

Keyword(s):

Germ Cells ◽

Primordial Germ Cells ◽

Cell Lineage ◽

Ventral Side ◽

Drosophila Embryo ◽

The Real ◽

Genital Disc ◽

Stage Of Development ◽

Data Support ◽

Blastoderm Stage

The primordial germ cells and the gonadal mesoderm were mapped in the Drosophila embryo by analyzing the patterns of mosaicism in ‘normal’ and ‘transformed’ gynandromorphs. Relative to the adult cuticular markers the germ cells map as the posterior moststructure, which coincides with their known location in the blastoderm embryo. These data support the hypothesis that the gynandromorph map reflects the real position of the pri-mordia in the embryo. Since after the blastoderm stage the primordial germ cells migrateanteriorly these data also indicate that the map in fact corresponds to the blastoderm stageand not to a later stage of development. The genital disc maps as a single median primordium anterior and ventral to the germ cells, the gonadal mesoderm is located anterior to the genital disc and also forms a single median primordium on the ventral side of the embryo. The primordia for the genital disc and the gonadal mesoderm are unusually large in size, which presumably reflects some indeterminacy of the cell lineage leading to an ‘expansion’ of the map.

Download Full-text

Functional requirement of gp130-mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells

Development ◽

10.1242/dev.122.4.1235 ◽

1996 ◽

Vol 122 (4) ◽

pp. 1235-1242 ◽

Cited By ~ 3

Author(s):

U. Koshimizu ◽

T. Taga ◽

M. Watanabe ◽

M. Saito ◽

Y. Shirayoshi ◽

...

Keyword(s):

Germ Cells ◽

Intracellular Signaling ◽

Primordial Germ Cells ◽

Cell Formation ◽

Embryonic Stem ◽

Oncostatin M ◽

Receptor Complexes ◽

Binding Subunit

Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse primordial germ cells (PGC) in culture. The receptor complex for LIF comprises LIF-binding subunit and non-binding signal transducer, gp130. The gp130 was originally identified as a signal-transducing subunit of interleukin (IL)-6 and later also found to be a functional component of receptor complexes for other LIF-related cytokines (oncostatin M [OSM], ciliary neurotrophic factor [CNTF] and IL-11). In this study, we have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. OSM was able to fully substitute for LIF; both cytokines promoted the proliferation of migratory PGC (mPGC) and enhanced the viability of postmigratory (colonizing) PGC (cPGC) when cultured on SI/SI4-m220 cells. Interestingly, IL-11 stimulated mPGC growth comparable to LIF and OSM, but did not affect cPGC survival. IL-6 and CNTF did not affect PGC. In addition, a combination of IL-6 and soluble IL-6 binding subunit (sIL-6R), which is known to activate intracellular signaling via gp130, fully reproduced the LIF action of PGC. Both in the presence and absence of LIF, addition of neutralizing antibody against gp130 in culture remarkably blocked cPGC survival. These results suggest a pivotal role of gp130 in PGC development, especially that it is indispensable for cPGC survival as comparable to the c-KIT-mediated action. We have further demonstrated that a combination of LIF with forskolin or retinoic acid, a potent mitogen for PGC, supported the proliferation of PGC, leading to propagation of the embryonic stem cell-like cells, termed embryonic germ (EG) cells. Since EG cells were also obtained by using OSM or the IL-6/sIL-6R complex in place of LIF, a significant contribution of gp130-mediated signaling in EG cell formation was further suggested.

Download Full-text